1. Field of the Invention
This invention concerns compositions and methods for targeted delivery of RNAi species, such as siRNA. Preferably, the targeted delivery complexes comprise an antibody, antibody fragment or other targeting molecule conjugated to a carrier molecule for siRNA, such as a protamine or a dendrimer. More preferably, the targeted delivery complexes are made by the dock-and-lock (DNL) technique. The targeted delivery complex may comprise one or more siRNA species for delivery to a targeted cell or tissue. Most preferably, delivery of siRNA is effective to treat a disease, syndrome or disorder, such as cancer, autoimmune disease, immune dysfunction (e.g., graft-versus-host disease or organ transplant rejection), inflammation, infectious disease, cardiovascular disease, endocrine or metabolic disease or neurodegenerative disease. For disease therapy, the antibody or other targeting molecule binds to a target antigen produced by a diseased cell or tissue or otherwise associated with the disease state. The DNL complex may comprise multiple copies of a carrier molecule to efficiently deliver therapeutic siRNA to a target cell. The antibody or other targeting molecule may be a rapidly internalizing species to facilitate intracellular uptake of siRNA.
2. Related Art
RNA interference (RNAi) is a naturally occurring regulatory mechanism for control of gene expression in cells (Fire et al., 1998, Nature 391:806-11). RNAi is mediated by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules that interact with the catalytic RISC component argonaute (Rand et al., 2005, Cell 123:621-29). Types of RNAi molecules include microRNA (miRNA) and small interfering RNA (siRNA). RNAi species can bind with messenger RNA (mRNA) through complementary base-pairing and inhibits gene expression by post-transcriptional gene silencing. Upon binding to a complementary mRNA species, RNAi induces cleavage of the mRNA molecule by the argonaute component of RISC. Among other characteristics, miRNA and siRNA differ in the degree of specificity for particular gene targets, with siRNA being relatively specific for a particular target gene and miRNA inhibiting translation of multiple mRNA species.
Therapeutic use of RNAi by inhibition of selected gene expression has been attempted for a variety of disease states, such as macular degeneration and respiratory syncytial virus infection (Sah, 2006, Life Sci 79:1773-80). It has been suggested that siRNA functions in host cell defenses against viral infection and siRNA has been widely examined as an approach to anti-viral therapy (see, e.g., Zhang et al., 2004, Nature Med 11:56-62; Novina et al., 2002, Nature Med 8:681-86; Palliser et al., 2006, Nature 439:89-94). The use of siRNA for cancer therapy has also been attempted. Fujii et al. (2006, Int J Oncol 29:541-48) transfected HPV positive cervical cancer cells with siRNA against HPV E6 and E7 and suppressed tumor growth. siRNA-mediated knockdown of metadherin expression in breast cancer cells was reported to inhibit experimental lung metastasis (Brown and Ruoslahti, 2004, Cancer Cell 5:365-74).
Difficulties with siRNA-based therapies have included poor cellular uptake of exogenous siRNA and potential off-target effects on other genes (see, e.g., Kim et al., 2009, Trends Molec Med 15:491-500). Jackson et al. (2003, Nat Biotechnol 21:635-37) designed different siRNAs targeting IGF-1R and MAPK14 and showed silencing of nontargeted genes containing as few as eleven contiguous identical nucleotides to the siRNA species.
Attempts have been made to provide targeted delivery of siRNA to reduce the potential for off-target toxicity. Song et al. (2005, Nat Biotechnol 23:709-17) used protamine-conjugated Fab fragments against HIV envelope protein to deliver siRNA to circulating cells. Schiffelers et al. (2004, Nucl Acids Res 32:e149) conjugated RGD peptides to nanoparticles to deliver anti-VEGF R2 siRNA to tumors and inhibited tumor angiogenesis and growth rate in nude mice. Dickerson et al. used nanogels functionalized with anti-EphA2 receptor peptides to chemosensitize ovarian cancer cells with siRNA against EGFR. Dendrimer-conjugated magnetic nanoparticles have been applied to the targeted delivery of antisense survivin oligodeoxynucleotides (Pan et al., 2007, Cancer Res 67:8156-63).
Despite recent progress, a need exists in the field for more effective means of targeted delivery of therapeutic siRNA, to increase cellular uptake and minimize siRNA-related toxic side effects (Kim et al., 2009).